Dietary selenium is incorporated into at least 25 human proteins as the amino acid selenocysteine (Sec). Sec incorporation in an elongating polypeptide represents a modification of the standard protein synthetic machinery in that it requires the utilization of a novel translation elongation factor (eEFSec), a selenocysteine insertion sequence (SECIS) element in the 3' untranslated region of selenoprotein mRNAs, and a novel SECIS binding protein termed SBP2. These factors act in concert to alter the coding potential of specific UGA codons by specifying the insertion of the Sec-specific tRNA, Sec-tRNA[Ser]Sec. The focus of this proposal is on SBP2 and its mechanism of action. To date, functional analyses have established that SBP2 is required for Sec incorporation, possesses specific SECIS element binding activity and also physically interacts with the ribosome. Structure/function analysis of SBP2 has shown that it is comprised of three distinct domain: a dispensable N-terminal domain with no known function, a central "functional domain" that is required for Sec incorporation but not SECIS element binding, and a C-terminal SECIS element binding domain containing an RNA binding motif found in the family of kink-turn binding proteins (e.g.ribosomal protein L7Ae). Using a combination of in vitro studies and cell-based assays, the experiments proposed are designed to decipher the structure/function relationships within the SBP2 subdomains and identify novel components of the Sec incorporation machinery using a three-tiered approach. First, we propose to precisely define the amino acids required for Sec incorporation in order to lay a solid foundation for structural studies. Second, we will develop assays to study the function of the SBP2 N-terminal domain in order to gain insight into its potential regulatory role in Sec incorporation. Third, we will identify components of the Sec incorporation complex (SIC)by assembling SBP2-centered and selenoprotein mRNA-centered complexes in mammalian cells followed by complex purification and identification. As a whole, this work will provide fundamental and essential information regarding the mechanism of Sec incorporation - an essential process that will be an important target for strategies designed to maximize the beneficial properties of selenoprotein function.